CN109362224B

CN109362224B - Neuromodulation techniques

Info

Publication number: CN109362224B
Application number: CN201780020897.6A
Authority: CN
Inventors: C·M·普莱奥; J·M·艾什; V·E·科特罗; M·E·马里诺
Original assignee: General Electric Co
Current assignee: General Electric Co
Priority date: 2016-04-04
Filing date: 2017-04-04
Publication date: 2022-09-13
Anticipated expiration: 2037-04-04
Also published as: EP3439736A1; JP6876067B2; US20190117977A1; JP2019513447A; CN109362224A; WO2017176776A1; US11040199B2; US20210299449A1

Abstract

The subject matter of the present disclosure relates generally to techniques for neuromodulating lymph tissue, including applying one or more energy pulses to neurons of a subject, e.g., via electrodes positioned to deliver sufficient energy to the neurons, to modulate immune function. For example, the adaptive immune reflex of a subject may be modulated via neuromodulation.

Description

Neuromodulation techniques

Cross Reference to Related Applications

Priority from U.S. provisional patent application No. 62/318,035 entitled "TECHNIQES FOR NEUROMODOLATION OF LYMPHATIC TISSUE", filed 2016, 4, 2016 (the entire contents OF which are incorporated herein by reference) and U.S. provisional patent application No. 62/325,828 entitled "TECHNIQES FOR NEUROMODOLATION OF LYMPHATIC TISSUE", filed 2016, 21, 4, 21, 2016 (the entire contents OF which are incorporated herein by reference).

Background

The subject matter disclosed herein relates generally to neuromodulation of lymphoid and immune-related tissues, and in particular, to techniques for differentially stimulating or modulating a physiological response in response to neuromodulation and using this response in particular embodiments to assess neuromodulation effectiveness.

Neuromodulation has been used to treat a variety of clinical conditions. For example, electrical stimulation at various locations along the spinal cord has been used to treat chronic back pain. Such treatment may be performed by an implantable device that periodically generates electrical energy that is applied to tissue to activate certain nerve fibers, which in turn may cause a reduction in pain sensation. In the case of spinal cord stimulation, the stimulation electrodes are typically positioned in the epidural space, while the pulse generator may be positioned slightly away from the electrodes, for example in the abdominal or hip region, but connected to the electrodes via leads. In other implementations, deep brain stimulation may be used to stimulate specific regions of the brain to treat dyskinesia, and the stimulation location may be guided by neuroimaging. Such central nervous system stimulation is usually directed to local nerve or brain cell function.

Peripheral neuromodulation may be relatively more challenging than targeting larger structures of the central nervous system. As the peripheral nerve extends outward, the size of the nerve bundle decreases. In addition, small peripheral nerve fibers can control a substantial portion of the surrounding tissue, which makes locating and targeting such nerves for neuromodulation relatively challenging. However, the peripheral nervous system innervates many different organ structures in the body, and may require targeting of certain peripheral nerves.

Disclosure of Invention

In one embodiment, a neuromodulation method is provided that includes applying one or more energy pulses to a neuron of a subject, e.g., via an electrode positioned to deliver sufficient energy to the neuron, to modulate an adaptive immune system neuroreflex, wherein neuromodulation results in a coordinated immune response on a portion of the immune/lymphatic system.

In an exemplary embodiment, a neuromodulation method is provided that includes applying one or more energy pulses to a neuron of a subject via an electrode positioned to deliver sufficient energy to the neuron to stimulate an adaptive immune system neuroreflex, wherein immune cell migration or outflow through a local lymphatic structure is differentially modulated in a coordinated manner in the lymphatic system.

In another embodiment, a neuromodulation method is provided which includes applying one or more energy pulses to a neuron of a subject via an electrode positioned to deliver sufficient energy to the neuron to modulate an adaptive immune system neural reflex, wherein immune cell fate and/or phenotype within a local lymphoid structure is differentially modulated in a coordinated manner in the lymphatic system.

In another embodiment, a neuromodulation method is provided, comprising applying one or more energy pulses to a neuron of a subject via an electrode positioned to deliver sufficient energy to the neuron to modulate an adaptive immune system neuroreflex, wherein cytokine secretion profiles of immune cells within or exiting a local lymphoid structure are differentially modulated in a coordinated manner in the lymphoid system.

In another embodiment, a neuromodulation method is provided, comprising applying one or more energy pulses to a neuron of a subject via an electrode positioned to deliver sufficient energy to the neuron to modulate an adaptive immune system neuroreflex, wherein checkpoint molecule expression of immune cells within or exiting a local lymphoid structure is differentially modulated in a coordinated manner in the lymphoid system.

In exemplary embodiments, modulation of adaptive immune system neuro-reflexes modulates immune function of local and systemic lymphoid tissue function through differential changes in one or more neurotransmitters or neuropeptides in the lymphoid tissue (or lymph fluid) in response to one or more energy pulses.

In an exemplary embodiment, a method of neuromodulating adaptive immune reflexes is provided, which includes applying one or more energy pulses to neurons of a subject to deliver sufficient energy to the neurons to neuromodulate lymphatic tissue in response to applying the one or more energy pulses.

In an exemplary embodiment, a method of neuromodulating adaptive immune reflexes is provided, comprising applying one or more energy pulses to a neuron of a subject via an electrode positioned to deliver sufficient energy to the neuron to modulate adaptive or innate immune function of contralateral lymphoid tissue such that a concentration or level of one or more neurotransmitters or neuropeptides in the contralateral lymphoid tissue or the contralateral lymphoid fluid is differentially altered in response to the one or more energy pulses.

In another embodiment, a method of neuromodulating adaptive immune reflexes is provided, comprising applying one or more energy pulses to neurons of a subject via electrodes positioned to deliver sufficient energy to the neurons to modulate immune function of lymphoid tissue such that the concentration of norepinephrine or epinephrine (epinephrine) in the lymphoid tissue or fluid increases by at least 100% relative to a pre-stimulation baseline in response to the one or more energy pulses, wherein the one or more energy pulses are applied at an energy ranging from 0.5V to 10V.

In another embodiment, a method of neuromodulating adaptive immune reflexes is provided, comprising applying one or more energy pulses to a neuron of a subject via an electrode positioned to deliver sufficient energy to the neuron to modulate immune function of lymphoid tissue such that the concentration or level of substance P in the lymphoid tissue or lymph fluid increases by at least 50% relative to a pre-stimulation baseline in response to the one or more energy pulses, wherein the one or more energy pulses are applied at an energy ranging from 0.5V to 10V.

In another embodiment, a method of neuromodulating adaptive immune reflexes is provided, comprising applying one or more energy pulses to a neuron of a subject via an electrode positioned to deliver sufficient energy to the neuron to modulate immune function of lymphoid tissue such that a concentration or level of vasoactive intestinal peptide in the lymphoid tissue or fluid increases by at least 50% relative to a pre-stimulation baseline in response to the one or more energy pulses, wherein the one or more energy pulses are applied at an energy ranging from 0.5V to 10V.

In another embodiment, a method of neuromodulating adaptive immune reflexes is provided, comprising applying one or more energy pulses to a neuron of a subject via an electrode positioned to deliver sufficient energy to the neuron to modulate immune function of lymphoid tissue such that the concentration or level of neuropeptide Y in the lymphoid tissue or lymph fluid increases by at least 100% relative to a pre-stimulation baseline in response to the one or more energy pulses, wherein the one or more energy pulses are applied at an energy ranging from 0.5V to 10V.

In another embodiment, a neuromodulation method is provided, including positioning an electrode at a location on or near a lymphoid tissue of a subject, where the electrode is capable of stimulating neurons innervating the lymphoid tissue; applying one or more energy pulses to the tissue via the electrodes to stimulate neurons to modulate lymphatic or immune function of the lymphatic tissue; assessing a state of lymphatic or immune function within the subject after applying a plurality of energy pulses based on a characteristic associated with lymphatic or immune function; and modifying a parameter of at least one of the plurality of energy pulses based on the state.

In another embodiment, a method for closed-loop neuromodulation is provided. The method comprises applying one or more energy pulses to the tissue to stimulate neurons to modulate lymphatic or immune function of the lymphatic tissue; and assessing the status of lymphatic or immune function within the subject after applying the plurality of energy pulses.

In another embodiment, a method for closed-loop neuromodulation is provided. The method comprises the following steps: controlling a pulse generator to apply one or more energy pulses to neurons innervating lymphatic tissue via the electrodes and in accordance with one or more parameters of at least one of the plurality of energy pulses to modulate lymphatic function of the lymphatic tissue; receiving information relating to a condition or function of lymphatic tissue; and changing one or more parameters based on the information.

In another embodiment, a method for neuromodulation is provided, the method comprising: receiving one or more user inputs selecting a mode of operation for delivering energy pulses to the electrodes to stimulate lymphatic tissue activity; delivering one or more energy pulses from a pulse generator to an electrode according to the mode of operation to cause stimulatory activity in lymphatic tissue; receiving one or more inputs related to the stimulatory activity of the lymphoid tissue; and changing the operation mode based on the input.

In another embodiment, a method for neuromodulation is provided. The method comprises the following steps: positioning an electrode at a location where the electrode is capable of stimulating neurons innervating lymphatic tissue; applying one or more energy pulses to the tissue via the electrodes to stimulate neurons to modulate lymphatic function of the lymphatic tissue; assessing the size of the lymphatic tissue relative to a baseline size after applying the plurality of energy pulses; and modifying a parameter of at least one of the plurality of energy pulses based on the size of the lymphatic tissue.

In another embodiment, a method of neuromodulating adaptive immune reflexes is provided, the method comprising applying one or more energy pulses to a neuron of a subject via an electrode positioned to deliver sufficient energy to the neuron to neuromodulate lymphoid tissue in response to applying the one or more energy pulses to promote entry or egress of cells from the lymphoid tissue or lymph fluid.

In another embodiment, a neuromodulation method is provided, comprising applying one or more energy pulses to neurons of a subject to modulate nerves that enter lymphatic tissue, wherein the applying results in an increase in tissue or blood levels of one or more endogenous opioids within the subject relative to a preconditioned control.

Drawings

These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

figure 1A is a schematic overlay of the central and peripheral nervous systems and lymphatic system (the picture shows secondary lymphoid structures and lymphatic vessels, but the system also includes primary lymphoid organs, such as the spleen);

FIG. 1B shows potential sites of electrical stimulation or neuromodulation on peripheral nerves located between the lymphatic system and the CNS;

FIG. 1C is a schematic representation of potential innervation of secondary lymphoid tissue (i.e., lymph nodes);

FIG. 2A is a schematic diagram of one of the simplest types of adaptive immune reflexes that can exist within the lymphatic system, including afferent neurons that control the neural output of efferent neurons that innervate specific regional lymph node compartments to modulate the adaptive immune process;

FIG. 2B shows an exemplary adaptive immune process with lymphocyte draining from the lymphatic system that will control the screening time of lymphocytes within lymph nodes;

FIG. 3 illustrates additional levels of adaptive immune control that may exist within the adaptive immune reflex via central prominent afferent neurons that may traverse multiple layers of the lymphatic system or spine and achieve coordinated regulation of lymphocyte drainage across multiple lymph nodes;

figure 4 shows additional levels of adaptive immune control that may be present within the adaptive immune reflex via central prominent afferent neurons that may traverse multiple levels of the spine of the lymphatic system to allow differential modulation of contralateral lymphoid structures, such as reduction of lymphocyte drainage at sites of inflammation/antigenic perturbation, except for migration of lymphocytes from lymph nodes in other distal lymphoid regions;

figure 5 shows additional levels of adaptive immune control that may be present within the adaptive immune reflex via inhibitory neurons that may exert spontaneous inhibition on efferent neurons, allowing for the timed separation of neural signals caused by initial inflammation/antigenic perturbation and where the inhibitory signals may be due to afferent signals from the same nerve, thereby causing an initial response within or outside the lymph node environment;

figure 6 illustrates additional levels of adaptive immune control that may be present within an adaptive immune reflex, wherein one neuron may exert mutual inhibition on efferent neurons by synapseing with both the efferent neuron and a second efferent neuron through inhibitory interneurons to allow differential modulation of immune activity within different lymphocytes, e.g., for reducing neural output to different parts of a lymph node;

FIG. 7 illustrates an additional level of adaptive immune control that may be present within the adaptive immune reflex, wherein signals from efferent neurons may be fed back through inhibitory interneurons onto an auto-inhibitory loop to allow self-regulation of the time and/or magnitude of the adaptive immune perturbation;

figure 8 shows how the different mechanisms of adaptive immune reflex can exert control in the local lymphatic network without interacting directly with the CNS or spinal column;

figure 9 shows additional levels of adaptive immune control that may be present within the adaptive immune reflex. In this case, the signals from the local reflections may be directly connected to a pathway extending through the brainstem and higher brain regions;

FIG. 10 shows an example of a number of regions within lymphoid tissue that may be affected by adaptive immune reflexes and a particular adaptive immune process that may be modulated by neuromodulation at each region;

fig. 11 shows an example of different areas of lymphatic drainage, where a specific lymph node is responsible for draining fluid from a specific area of the body;

FIG. 12 shows an experimental procedure for obtaining the ability to differentially stimulate an adaptive immune reflex;

figure 13 shows an image of a resected popliteal lymph node on a stimulated leg and a contralateral/unstimulated leg;

FIG. 14 shows the results of comparing the weight of the stimulated and unstimulated popliteal lymph nodes;

FIG. 15A shows the concentration of epinephrine in the popliteal lymph node (stimulated leg) after stimulation with different voltages;

figure 15B shows the concentration of epinephrine in lymph fluid after stimulation with different voltages;

FIG. 16A shows the concentration of norepinephrine in the popliteal lymph node after stimulation with different voltages;

figure 16B shows the concentration of noradrenaline in lymph fluid after stimulation with different voltages;

figure 17A shows the concentration of dopamine in the popliteal lymph nodes after stimulation with different voltages;

fig. 17B shows the concentration of dopamine in lymph fluid after stimulation with different voltages;

FIG. 18A shows the concentration of neuropeptide Y in the popliteal lymph node after stimulation with different voltages;

figure 18B shows the concentration of neuropeptide Y in lymph fluid following stimulation with different voltages;

FIG. 19A shows the concentration of substance P in the popliteal lymph node after stimulation with different voltages;

FIG. 19B shows the concentration of substance P in lymph fluid after stimulation with different voltages;

FIG. 20A shows the concentration of vasoactive intestinal peptide in the popliteal lymph node following stimulation with different voltages;

figure 20B shows the concentration of vasoactive intestinal peptide in lymph fluid following stimulation with different voltages;

FIG. 21 shows a comparison of adrenal hormone (adrenaline) concentrations in stimulated and unstimulated contralateral lymph nodes from the same subject;

FIG. 22 shows a comparison of dopamine concentrations in stimulated and non-stimulated contralateral lymph nodes in the same subject;

FIG. 23A shows the concentration of epinephrine in the lymphatic tissues of stimulated lymph nodes with intact nerves, stimulated lymph nodes with severed nerves, and controls;

FIG. 23B shows the concentration of norepinephrine in lymphatic tissues of stimulated lymph nodes with intact nerves, stimulated lymph nodes with severed nerves, and controls;

figure 23C shows the concentration of dopamine in lymphoid tissue of stimulated lymph nodes with intact nerves, stimulated lymph nodes with severed nerves, and controls;

FIG. 23D shows the concentration of neuropeptide Y in lymphoid tissue of stimulated lymph nodes with intact nerves, stimulated lymph nodes with severed nerves, and controls;

FIG. 23E shows the concentration of substance P in lymphoid tissue of stimulated lymph nodes with intact nerves, stimulated lymph nodes with severed nerves, and controls;

FIG. 23F shows the concentration of vasoactive intestinal peptide in lymph tissue of a stimulated lymph node with intact nerves, a stimulated lymph node with severed nerves, and a control;

FIG. 24A shows the concentration of epinephrine in lymph nodes directly stimulated at different stimulation frequencies (applied voltage was maintained at 0.5V);

FIG. 24B shows the concentration of norepinephrine in lymph nodes directly stimulated at different stimulation frequencies (applied voltage was maintained at 0.5V);

fig. 24C shows the concentration of dopamine in lymph nodes directly stimulated at different stimulation frequencies (applied voltage was kept at 0.5V);

FIG. 24D shows the concentration of epinephrine in lymph taken after direct stimulation of lymph nodes at different stimulation frequencies (applied voltage was maintained at 0.5V);

FIG. 24E shows the concentration of norepinephrine in the uptake lymph fluid following direct stimulation of the lymph nodes at different stimulation frequencies (applied voltage was maintained at 0.5V);

figure 24F shows the concentration of dopamine in the lymph fluid taken after direct stimulation of the lymph nodes at different stimulation frequencies (applied voltage was maintained at 0.5V);

FIG. 25A shows the concentration of neuropeptide Y in lymph nodes directly stimulated at different stimulation frequencies (applied voltage was maintained at 0.5V);

FIG. 25B shows the concentration of substance P in lymph nodes directly stimulated at different stimulation frequencies (applied voltage was kept at 0.5V);

FIG. 25C shows the concentration of vasoactive intestinal peptide in lymph nodes directly stimulated at different stimulation frequencies (applied voltage was kept at 0.5V);

FIG. 25D shows the concentration of neuropeptide Y in the lymph taken up after direct stimulation of lymph nodes at different stimulation frequencies (applied voltage was kept at 0.5V);

FIG. 25E shows the concentration of substance P in lymph taken after direct stimulation of lymph nodes at different stimulation frequencies (applied voltage maintained at 0.5V);

FIG. 25F shows the concentration of vasoactive intestinal peptide in the lymph taken up after direct stimulation of the lymph nodes at different stimulation frequencies (applied voltage was kept at 0.5V);

FIG. 26 is a comparative graph showing the number of immune cells in lymph nodes excised from stimulated, sham and initial animals after dissection and dissociation into single cell suspensions;

fig. 27 is a comparative graph showing the number of immune cells per microliter in collected lymph or blood of the same subject as in fig. 26;

fig. 28 is a comparative graph showing the number of immune cells per microliter of dissociated samples from various primary immune organs of the same subject as fig. 26 and 27;

FIG. 29 shows stimulation results based on differential stimulation of electrode placement, where the gap between the electrode and the nerve results in firing of only a subset of axons;

figure 30 shows stimulation of lymphocyte uptake in the liver and experimental results for the same subject/experiment of figure 29;

FIG. 31 shows the stimulation of lymphocyte uptake in the spleen and the results of the experiment for the same subject/experiment in FIG. 29;

FIG. 32 is a schematic diagram of a neuromodulation system using electrodes positioned on a nerve, according to embodiments of the present disclosure;

fig. 33 is a block diagram of the system of fig. 32, according to an embodiment of the present disclosure;

figure 34 is a flow diagram of a neuromodulation and monitoring technique according to embodiments of the present disclosure;

figure 35 is a schematic diagram of a neuromodulation system for immunomodulation, according to embodiments of the present disclosure;

FIG. 36 is an image of a stimulated popliteal lymph node labeled with a dye for visualization;

FIG. 37 is an ultrasound image before the stimulated popliteal lymph node was stimulated, with embedded length measurements of the lymph nodes;

FIG. 38 is an ultrasound image after the stimulated popliteal lymph node has been stimulated, with embedded length measurements of the lymph nodes;

FIG. 39 is a calibration chart of a competition assay for the fluorescent detection of β endorphin; and

fig. 40 is a graph showing test results for stimulated subjects relative to unstimulated controls.

Detailed Description

One or more specific embodiments will be described below. In an effort to provide a concise description of these embodiments, not all features of an actual implementation are described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

Any examples or illustrations given herein are not to be considered in any way as limitations, restrictions, or express definitions of any term or terms used in connection with them. Rather, these examples or illustrations should be considered in relation to various specific embodiments and are by way of illustration only. Those of ordinary skill in the art will understand that any one or more of the terms used in connection with these examples or illustrations will include other embodiments that may or may not be given together or elsewhere in the specification, and all such embodiments are intended to be included within the scope of the term or terms. Language designating such non-limiting examples and illustrations includes, but is not limited to: "for example," such as, "" like, "" including, "and" in one embodiment.

The present technology relates to the differential neuromodulation of adaptive immune reflexes to cause activation of cell-or tissue-based physiological effects in lymphoid and peripheral tissues. For example, immune cells are known to respond to chemical stimuli, such as neurotransmitters/neuropeptides released by nerves (released in response to action potentials and bioelectrical activity). However, the extent to which these stimuli modulate immune function, as compared to phenotypic cytokines and inflammatory signals, remains unknown. In addition, the location and tissue microenvironment where these neuro-immune signals affect the greatest possible is unknown. Although the rough anatomy of the innervation in lymphoid tissues is known, the functional role these nerves play in immune cell and tissue function has not been determined. A number of pacemakers of peripheral nerves innervating lymphatic vessels, as well as electrical stimulation, are shown to alter lymphatic flow in a manner related to vasoconstriction/vasodilation in blood vessels. However, the functional consequences of neurotransmitter and neuropeptides released on immune cells in the lymphatic system and lymphoid tissues/organs (including cortical and accessory cortical regions involved in B and T cell-related activities) have not been determined. Most importantly, the ability of the nervous system to arrange an adaptive immune response on the neural network that innervates multiple lymphoid tissues/organs, or the ability of researchers to differentially modulate this effect, has not been demonstrated.

Vagal stimulation has been used to stimulate the cholinergic anti-inflammatory pathway. In this pathway, the efferent arms of the vagus nerve innervate the celiac ganglia, with the splenic nerve protruding into the spleen. This splenic protrusion releases acetylcholine within the spleen, which interacts with nicotinic AChR on macrophages (and other cytokine-producing cells). This interaction inhibits TNF (and other proinflammatory cytokines) production in the spleen, thereby producing a systemic anti-inflammatory response by modulating the innate immune system.

However, the immune system is complex, and neuroreflexes may not only regulate systemic molecular expression associated with innate immunity. Lymph nodes and lymphoid tissues represent the main physiological targets for studying neuro-immune regulation. Lymphoid tissue is the primary site for control of adaptive immunity because antigen-presenting cells (e.g., dendritic cells) home to the lymph upon activation of the antigen, while primary lymphocytes home to the lymph during inflammation. These two mechanisms serve to significantly increase the chance of the naive lymphocytes interacting with their cognate antigen. These cell migration or homing processes can be activated by changes in immune cell phenotype (i.e., upregulation of proteins associated with endosmosis and extravasation), phenotype associated with lymphatic or vascular endothelium at the lymph node inlet (i.e., surface proteins associated with immune cell endothelial interactions or endothelial layer permeability), or phenotype associated with lymphatic endothelium at the lymph node outlet. These cell homing processes can also be altered by regulating the general flow of lymphatic fluid. In fact, one of the remaining open questions in immunobiology is how some memory cells respond significantly and rapidly (and possibly several meters away from the nearest memory cell) to antigens present only in the local tissue environment.

In addition to immune cell migration, the microenvironment of the lymphatic compartment is associated with cell fate upon homologous antigen binding and activation. In general, microenvironments enriched in "inflammation-associated" proteins and signaling molecules drive activated lymphocytes or immune cells into an "effector phenotype". Effector cells typically express proteins associated with "fighting infection or foreign invaders". For example, effector T cells include cytotoxic T cells or helper T cells capable of inducing B cell maturation or macrophage activation. In general, microenvironments rich in "anti-inflammatory" proteins and signaling molecules produce an "inhibitory phenotype". Suppressor cells typically express proteins associated with "suppressing an active immune response". For example, suppressor T cells are regulatory T cells that help reduce excessive immune responses to "self-antigens" (i.e., maintain immune tolerance). These examples represent only a few immune cell (and supporting cell) phenotypes that depend on the dynamic lymphoid microenvironment state.

Lymph nodes (and lymph-related tissues) are innervated by neurons; early studies revealed a wide subset of neurons within the lymphatic compartment, including staining of neurons positive for a variety of neurotransmitter and neuropeptide receptors. In addition, lymph nodes are structured into distinct immune functions, which are divided into specific tissue structures. Barrier tissues (such as high endothelial venules) are used to regulate the entry/exit rate of immune cells from lymphoid tissues. The subcortical and cortical regions accommodate T cells, while the central follicle accommodates B cells. The supporting cells include: a fibrous structure that allows cells to migrate between "zones," and resident antigen presenting cells that can further regulate lymphocyte function and trafficking. The primary lymphocytes and cells regularly pass through these areas during their circulation from the tissue through the lymphatic and circulatory systems. Activated cells respond to migratory signals (originating from surrounding cells) and move from one lymphocyte to the next to perform their function. For example, B cells activated and proliferating in the follicular region migrate to the marginal zone (between the T cell-rich and B cell-rich regions of the lymph). In the limbic region, B cells can interact with helper T cells, which provide the signals necessary for full maturation to plasma cells (i.e., antibody-producing cells); mature plasma cells then migrate to the efferent lymphatic and medullary sinus structures and begin to produce antibodies that are secreted into the circulation. Although innervation of these structures is known; little is known about the functional significance of neural signaling in these local lymphatic microenvironments. Although neurotransmitter and neuropeptide receptors have been shown to be present at very specific locations within lymph node structures/chambers, there is no evidence that those receptors will be used to transmit information back to the central nervous system (i.e., under certain conditions, receptors may only be used to locally release neurotransmitters/neuropeptides, which act (like cytokines) as local immunomodulators). Still, the presence of functional neuro-reflex circuits (i.e. fast/local reflex between the lymphatic compartment and the central nervous system and/or slow/global circuits between the lymphatic compartment and the central nervous system) will have profound effects on the study and therapeutic applications of neuro-immune interactions.

To date, several studies have shown that in vivo injection of molecules commonly classified as neurotransmitters or neuropeptides can modulate local immune activity. For example, noradrenaline is thought to alter lymphocyte emigration in the lymphatic system, and VIP is thought to induce regulatory functions/phenotypes in T cells. However, these functional results have not been demonstrated via direct nerve stimulation (where release profile and concentration are controlled by nerve location and synaptic activity), but rather via systemic injection of high concentrations of nerve signaling molecules to demonstrate immunomodulation. Thus, the importance of neural activity in locally modulating these immune processes remains unknown. More importantly, since the investigation relies on varying concentrations of neural signaling molecules on a large scale or a systemic scale, it is not known whether local neural reflexes can provide a coordinated or synergistic response to immune modulation (via distant neural signaling between lymphoid tissues). Thus, the network of afferent and efferent neurons may still provide a distant coordinated response mechanism to local immune damage on the lymph structure. These neuroreflexes can modulate immune activity in multiple length dimensions and be therapeutically exploited by specific differential stimulation.

The spinal motion system represents a good analog system for postulating and testing various neural reflexes that may exist in immune or lymphoid tissues. Neuroreflex circuits within the spinal motor system allow the CNS to effectively measure muscle strength and length (and thus provide the correct motor response to the desired perturbation). Sensory or afferent neurons within the muscle spindle are capable of firing alpha motor neuron activity. The golgi tendon afferent nerves are capable of inhibiting (via interneurons in the spine) alpha motor neuron activity. In addition, the gamma motor neuron is able to control the length of the muscle spindle itself and thus set the sensitivity of the muscle spindle afferent. These neuroreflexes are responsible for controlling the standard hammer reflex of the knee tendon, where tapping the knee tendon results in stretching of the muscle sensory afferent nerves and coordinated firing/relaxation of the extensors and flexors of the leg, respectively (where the inhibitory spinal interneurons are responsible for the opposite flexor/extensor responses to the perturbation). It is speculated that similar (but not yet known) reflexes associated with immunization and neuro-immunomodulation of lymphoid tissues may be present, since the neuronal synapses are present within lymphoid tissues and the neuronal signaling molecules appear to alter the immunological activity. Furthermore, it is understood that this circuit may allow differential modulation of the adaptive immune response on the lymphatic/immune tissue network.

Continuing with motor neuron simulation, it is also known that cortical neurons can directly alter motor neuron responses to standard muscle reflexes. In this case, active consideration is given to changing the latency of the response of the muscle to stretching against the stretching or perturbation of the muscle (before stretching occurs). This is due to the presence of the second motion reflection path. The spinal reflexes discussed above are referred to as "short latency response pathways" and such other reflexes that rely on cortical neural activity are referred to as "long latency response pathways". Afferent inputs (from muscle spindles) associated with the long latency pathway must pass through the dorsal column nucleus and additional interneurons in the thalamus and thus provide a reflex with a delayed response (or latency) compared to the spinal reflex. The result of these two pathways is the presence of long term "voluntary reactions" and short latency "involuntary reactions" associated with disturbances in muscle tone. Furthermore, it can be speculated that since the neuronal synapses are present with lymphoid tissue and the neural signaling molecules appear to alter immune activity, there may be similar (but not yet known) cortical reflexes or neural pathways that provide control over the neural immunomodulation in immune and lymphoid tissues. Furthermore, understanding this circuit may allow for differential modulation of adaptive immune responses in the lymphatic/immune tissue network, as well as additional information about cortical control (such as the likelihood of "immunological memory" or information stored in the cortex about specific inflammatory or antigenic perturbation events).

None of the current findings suggest the ability of the nervous system to coordinate adaptive immune responses to local immune perturbations and/or lesions. Systemic neuroimmune reflexes, such as cholinergic anti-inflammatory pathways, have been discovered and applied. However, these pathways are involved in global/systemic control of the innate immune response and are not the mechanism for generating a coordinated adaptive immune response throughout the immune and lymphoid tissue systems in vivo. The disclosure and application of such locally adaptive immune reflexes will have profound effects on basic science and transformation medicine.

As discussed, lymph nodes are compact immune tissues divided into distinct chambers, each containing a different subset of immune cells with different immune functions. In addition, these chambers are innervated by different types of neurons. However, the neural signals (action potential frequency, stimulation intensity) required to cause functional changes in the state of immune cells are unknown. Certain innervated lymph node compartments include regions innervated by T cells, where dendritic cells migrate and present antigen to the cells for activation. Innervation also follows blood vessels, some of which are responsible for controlling migration/entry of immune cells from peripheral tissues. For example, lymphocytes in the bloodstream may enter lymph nodes through the High Endothelial Venules (HEV). In addition, the rate at which lymphocytes and fluids enter and leave the lymph nodes affects the volume of the lymph nodes. Within the lymph nodes, supporting cells and tissues play a role in regulating immune cell responses to self-antigens and maintaining immune cell tolerance. Other microenvironments within the lymph node, once activated by specific cognate antigens, affect the phenotype of immune cells, e.g., endothelial cells within the lymph node outlet vessel provide a fixed location and microenvironment for activated plasma cells that release antibodies into the bloodstream. As an additional effect, the lymph nodes and lymphatic system serve to transport, filter and drain lymph fluid produced from interstitial fluid of upstream tissues. For example, it is known that the protein composition of the efferent lymph fluid of a lymph node is generally higher than its afferent lymph fluid. The flow of afferent and efferent lymph of the lymph nodes is regulated and involves contraction of the smooth muscle cells of the lymphatic vessels (0.6 to 10 times per minute), lymphatic structure, and even contraction of the lymphatic smooth muscle around the outer wall of the lymph nodes (0.5 to 1 times per minute). Neuromodulation of lymphoid tissue may alter the drainage rate of the drainage fluid and/or the cell population in the drainage fluid. These (and other) adaptive immune processes occurring in lymphoid tissues are associated with a number of diseases, including infection, inflammatory/autoimmune disease, allergy and immune reactions to tumors or foreign bodies (grafts and implants). In one embodiment of the disclosed technology, local stimulation of specific nerves upstream of, adjacent to, or within lymphatic tissue results in 1) targeted downstream immunomodulation within the lymphatic tissue and 2) orchestrated or consistent immunomodulation in the adjacent lymphatic tissue by adaptive immune reflex pathways. Provided herein are techniques for differential immunomodulation in the lymphatic network based on stimulation of adaptive immune reflexes.

As provided herein, neuromodulation or neuromodulation of a subject refers to applying energy to a neuron or nerve via an introduced energy source, which may be an internal or implanted energy source or an energy source external to the subject. The energy source may be a pulse generator that generates electrical or other energy pulses that are applied to the nerve via one or more electrodes. Neuromodulation (i.e., performing neuromodulation) may include nerve stimulation, or application of energy to activate or increase a nerve or nerve function. Neuromodulation may also include blocking or reducing energy application to a nerve or nerve function. It should be understood that neuromodulation may be achieved by additional or alternative techniques. However, in the context of the present disclosure, neuromodulation is achieved at least via application of energy via one or more electrodes positioned such that application of an energy pulse modulates a neuron or nerve at a desired location. In particular embodiments, the electrodes may be positioned within the lymphatic tissue, proximate to (e.g., on or near) the nerves innervating the lymphatic tissue, or at a location on the external surface of the subject's skin (or mucosal tissue) such that the applied energy transdermally activates the neurons or nerves.

To this end, the disclosed neuromodulation techniques may be used to locally or differentially modulate an adaptive immune reflex. FIG. 1A is a schematic or overlay of the central nervous system, peripheral nervous system, and lymphatic system. Adaptive immune responses in the neural pathway facilitate coordinated or orchestrated regulation of adaptive immune responses throughout the lymphatic system via neural signaling. Neuromodulation of an adaptive immune response may be performed on any nerve that innervates a particular lymphoid tissue (or lymph node, as shown in fig. 1B), wherein the adaptive immune response may be modulated to achieve an immune response or result that is different from that of the surrounding tissue. Stimulation may include stimulation of sensory/afferent and/or efferent/effector nerve fibers. The site of modulation may be any suitable neural site that modulates the immune and/or lymphatic responses provided.

FIG. 1B is a schematic illustration of what is known about the innervation of the lymph node environment. Different nerve types (including sympathetic or catecholamine neurons and neuropeptide neurons) have been observed at different locations within lymph node structures. These innervate the lymph nodes, as shown in FIG. 1C, at the apex (posterior to the blood vessel), and terminate in various regions of the lymph nodes, including the subcortical, interfollicular regions, and the medullary sinus. These nerves travel along the lymphatic vessels and also innervate blood and lymphatic vessels that connect the lymph nodes within the lymphatic system.

In certain embodiments, neuromodulation will modulate the adaptive immune reflex. Adaptive immune reflexes may involve afferent neuronal activity due to inflammation or the presence of antigens to prevent lymphocyte discharge from lymph nodes. An increase in lymphocytes in the lymph nodes may increase antigen screening. Figure 2A shows a schematic of one potential pathway of adaptive immune reflex. In this simple pathway example, afferent neurons (e.g., sensory neurons) in lymphatic or peripheral tissues are transmitted to efferent neurons that innervate specific locations within lymph node structures. This may be, for example, the medulla and endothelium in the interfollicular region, which is responsible for the gated draining of lymphocytes from lymph nodes. Signaling from afferent neurons (modulated by inflammatory molecules or antigens) can affect the firing of such efferent neurons, and alter the local concentration of neurotransmitters or neuropeptides (which can modulate the rate of lymphocyte drainage through the endothelial barrier, e.g., by changing permeability or by affecting the exit sinusoid). Figure 2B is a schematic representation of altering the permeability of the endothelial barrier to lymphocytes to allow passage across the barrier. This adaptive immune reflex may be important to allow a rapid reduction of lymphocyte shedding upon infection, which may rapidly increase lymphocyte screening of antigen from antigen presenting cells migrating to the lymphoid compartment.

Figure 3 shows another exemplary neural pathway of an adaptive immune reflex, in which afferent neurons (signaling inflammation or the presence of an antigen) continue into and up the central nervous system. In this case, neural signaling from the nerve may be used to provide a coordinated or orchestrated adaptive immune response by signaling multiple efferent neurons, innervating multiple lymph nodes or lymphoid tissues in the network. For example, the draining of lymphocytes from lymph nodes throughout the entire portion of the lymphatic system may be reduced due to the presence of a single local perturbation or stimulus, such as a local inflammatory or infectious event. Afferent nerve inputs to the adaptive immune reflex may travel up the spine to coordinate the immune reflex throughout the lymphatic region, e.g., stimulation of the foot may alter lymphocyte discharge throughout the leg.

Figure 4 shows another exemplary neural pathway of an adaptive immune reflex, in which afferent neurons (signaling inflammation or the presence of an antigen) continue into and up the central nervous system. In this case, neural signaling from the nerve can be used to provide opposite adaptive immune results at different locations within the lymphatic system. For example, lymphocyte depletion in the lymphatic system surrounding local inflammation or antigen injury may be reduced (to allow rapid or increased lymphocyte screening in those lymph nodes), but lymphocyte depletion in distant parts of the lymphatic system may be increased in order to move lymphocytes to the infected area. As shown, this type of effect may be produced by interneurons that fire effector neurons in one part of the lymphatic system, but inhibit efferent neurons innervating other/distant areas.

Figure 5 shows another exemplary neural pathway of an adaptive immune reflex, in which afferent neurons (signaling inflammation or the presence of an antigen) excite inhibitory neurons, which reduce the activity of efferent nerves in the adaptive immune reflex. This auto-suppression can be used in conjunction with the simple neural pathway described in fig. 2A to provide feedback or inverse neural signals to counteract the initial immune outcome (as shown by using lymphocyte depletion as an example). As shown, afferent signaling may originate from tissue activity in lymph or surrounding tissue (thus conveying information about the adaptive immune response at various stages). After a long-term inflammatory response or local changes in the lymph node environment (i.e., cognate antigen recognition and lymphocyte proliferation), different afferent/sensory neurons may form an anti-synaptic reflex, where inhibitory neurons serve to shut down the initial response and again allow lymphocytes to be drained from the lymph nodes. This will allow a feedback control mechanism for the initial reaction.

Figure 6 shows another exemplary neural pathway of an adaptive immune reflex, in which an afferent neuron (signaling inflammation or the presence of an antigen) excites a neural pathway associated with two different efferent neurons. Inhibitory neurons can exist between afferent nerves and an efferent pathway, allowing for reciprocal inhibition of adaptive immune responses and/or differential neuromodulation at different locations within lymphatic tissues/lymph nodes. The cross inhibitory reflex by unilateral inhibition of neurons can be used to reduce neural output to different parts of the lymph nodes. For example, in fig. 6, efferent 1 may be used to reduce lymphocyte trafficking, while efferent 2 may reduce neurotransmitter levels in the hair follicle or germinal center, thus promoting a more efficient immune cell phenotype upon cognate antigen recognition.

Figure 7 shows another exemplary neural pathway of adaptive immune reflex, in which efferent neural signals conduct feedback onto inhibitory neurons and achieve self-inhibition during neural firing. This configuration of reflex pathways will allow self-regulation, where efferent nerve excitation will feed back on itself to limit the duration and/or magnitude of neural signals in lymphatic tissues/lymph nodes. Specific suppressor cells in the spine can suppress the very same efferent neurons that are firing, thereby providing self-regulation. For example, the initial decrease in lymphocyte output is turned off after a period of time due to negative feedback from inhibitory neurons.

Figure 8 shows another example of an adaptive immune reflex neural pathway, wherein the neural pathway is completely contained within the peripheral nervous system (and does not include neurons within the spinal column or CNS). These neurons may be associated with excitatory and/or inhibitory neurons associated only with lymphatic or peripheral tissues, and may be connected by nerves that travel with lymphatic vessels. This neural network may contribute to the reflexes discussed above (i.e., reciprocal, autologous, intersecting, coordinated, and periodic reflex loops), and only pass within the neural network around the peripheral lymphoid tissue. The network of afferent/efferent neurons may synapse directly in lymphatic tissue and in lymphatic vessels and enable neuronal signaling between adjacent lymph nodes along lymph specific pathways.

Figure 9 shows another example of an adaptive immune reflex neural pathway, wherein the neural pathway is prominent throughout the CNS (including neurons with higher brain stem or cortical pathways). This type of long loop reflex may enable suppression of the portion of the local adaptive immune reflex through central nerve signaling. For example, as depicted, signals of systemic inflammation of the brainstem or higher ganglion neurons may be signaled to inhibit the cross-mobilization reflex described in fig. 4. This would allow mobilization of lymphocytes from distant lymphoid tissues to local infection under standard conditions, but eliminate this mobilization when the body is fighting systemic infection or injury. An additional effect of higher CNS inputs on the adaptive immune reflex would be to engage "immunological memory" or the cortex in coordinating and scheduling the response to specific inflammation or antigenic stimulation. As shown, the lesion memory may be mediated by signals representing systemic inflammation as a result of long-loop adaptive immune reflexes. Higher order reflections may allow for suppression of portions of the reaction. Longer cortical or CNS rings may promote the coordination of local immune reflexes of systemic physiological conditions. For example, cortical or CNS rings signaling chronic or systemic inflammation may inhibit the cross-mobilization reflex. As provided herein, neuromodulation may result in such local inhibition to treat systemic inflammation, or may be used to mobilize previously inhibited local responses.

Figure 10 depicts how nerve endings in different locations within the lymphoid tissue/lymph node structure can have significantly different effects on the adaptive immune system. Innervation of the high endothelial vessels may effect neuromodulation of lymphocyte flow from the blood into the lymphatic compartment, e.g., depending on the stimulation or blocking frequency used, neuromodulation may facilitate drainage or entry. Nerves within the capsule and cortex can effect neuromodulation of processes that control the entry of antigen presenting cells or the permeability of the reticulocyte network that filters lymph fluid. Innervation of the follicular region may effect, upon activation, modulation of B cell homing, B cell interaction with antigen presenting cells, or B cell phenotype. Nerves within the deep cortical unit may, upon activation, allow for the modulation of T cell homing, T cell interaction with antigen presenting cells, or T cell phenotype. Innervation of the interfollicular region may allow for modulation of T cell/B cell interactions, including the involvement of T cells in the growth of activated B cells into antibody-producing plasma cells. Nerves within the medullary region can effect neuromodulation of the discharge of lymphocytes and/or antigen presenting cells from lymph nodes, and/or modulate the phenotype of cells within the region (such as immobilized plasma cells).

Fig. 11 illustrates a known separation of the lymphatic system into multiple tissue drainage segments. Neuromodulation of specific portions of the adaptive immune reflex may allow for specific modulation of the adaptive immune process within localized regions of the lymphatic system.

FIG. 12 depicts a standard experimental method for studying differential stimulation or modulation of adaptive immune reflexes in one embodiment of the present invention. The dye is first injected into the footpad of the subject, which causes the dye to diffuse within the lymphatic system of the injected leg. This dye enables visualization of lymph nodes (lymph nodes not dissected in stimulation experiments) and nerves innervating selected lymph nodes. In one embodiment, the right popliteal lymph node is selected for direct stimulation, and the sciatic nerve is selected as the stimulation site directly above this lymph node. Bipolar insulated electrodes were placed around the sciatic nerve at this site (one pole on each side of the nerve) and stimulation was achieved using a power source and functional generator attached to leads in contact with both electrodes. Five minutes after stimulation, regional lymph nodes (along with other nodes and tissue compartments) were dissected, lymph fluid was collected via lymphatic vessels, and blood was collected from the great veins. Extracted tissues were processed and analyzed for neurotransmitter concentration (using HPLC or Elisa analysis), neuropeptide concentration (using Elisa analysis), or total specific immune cell count (inserted into hematology analyzer details; after further processing into single cell suspensions). Controls for these experiments included initial (no stimulation), sham (electrode insertion but no stimulation) and neurosectomy (cutting of the nerves that innervate the lymph nodes prior to stimulation) controls.

Lymphoid tissue

The present technology relates to the stimulation of immune cell and/or lymphatic system structures. As disclosed herein, stimulation can affect the function of the lymphatic system, such as cell accumulation, drainage, cell proliferation, and the like. The lymphatic system includes lymphoid organs, lymphatic vessels that extend throughout the body and provide flow and drainage, and lymphatic fluid that is transported within the lymphatic system. Lymphatic vessels transport immune cells from other tissues to lymph nodes and lymphoid organs, such as the spleen and thymus. Lymphatic vessels are a network of blood vessels that carry lymph fluid and cells and extend throughout the body into tissues. Primary lymphoid organs include the thymus and bone marrow. The spleen, lymph nodes, peyer's patches and collateral lymphoid tissue (including the tonsils and appendices) are secondary lymphoid organs. These organs consist of a scaffold of connective tissue that supports the circulation of B-and T-lymphocytes and other immune cells including, for example, macrophages, dendritic cells, and eosinophils. When microorganisms invade the body or the body encounters other antigens, the antigens are usually transported from the tissue to the lymph. Lymph is carried in lymphatic vessels to regional lymph nodes. In the lymph nodes, macrophages and dendritic cells phagocytose the antigen, process the antigen, and present the antigen to lymphocytes, which can then begin to produce antibodies or serve as memory cells to recognize the antigen again in the future. Thus, lymph and lymphoid tissues contain antibodies and immune cells. Lymphoid tissue is composed of well-defined structural components including cortex (e.g., primarily B lymphocyte filled regions, including follicles), paracortical (e.g., primarily T lymphocyte filled regions), and the lymphatic sinuses surrounded by regions normally filled by macrophages.

The lymph nodes act as filters for lymph fluid carried via the lymphatic vessels and include internal chambers of lymphocytes to collect and destroy bacteria and viruses carried via the lymphatic vessels. Lymph nodes also produce lymphocytes and antibodies. When the body is resistant to infection, these lymphocytes rapidly multiply and produce the characteristic swelling of the lymph nodes. Approximately 250 hundred million different lymphocytes migrate through each lymph node each day. Lymph is transported to progressively larger lymph vessels, finally to the right lymph vessel (for lymph of the right upper body) and the thoracic duct (for the rest of the body). These tubes drain into the circulatory system at the left and right subclavian veins near the shoulders. Along the lymphatic network is a series of various lymphoid tissues and organs including lymph nodes, peyer's patches, tonsils, lymph nodes, thymus and spleen. The lymph nodes encapsulate many lymph nodules within a tough sac and are provided with blood and lymph vessels. Lymph nodes filter the lymph transported to them by the lymphatic vessels. Thus, the lymph nodes filter the lymph draining from the lymphatic capillary bed in which the lymph node is located.

Lymph node clusters are found in various anatomical regions, and the methods of the invention can be used to locally neuromodulate one or more of these lymph regions. For example, lymph node clusters are found in the axilla (axillary lymph nodes), inguinal (inguinal lymph node), neck (cervical lymph node), chest (pectoral lymph node), and abdomen (iliac lymph node). Other lymphatic clusters include, but are not limited to, the popliteal lymph node, parasternal lymph node, lateral aortic lymph node, ventral aortic lymph node, submental lymph node, parotid lymph node, submandibular lymph node, intercostal lymph node, diaphragm lymph node, pancreatic lymph node, chylomicron (cisterna), lumbar lymph node, sacral lymph node, obturator lymph node, mesenteric lymph node, gastric lymph node, hepatic lymph node, and spleen lymph node. The disclosed techniques can be used to directly neuromodulate nerves innervating any one or more of these lymphatic regions.

Neuromodulation

The human nervous system is a complex network of nerve cells or neurons that is found in the center of the brain and spinal cord, as well as in the periphery of various nerves in the body. Neurons have a cell body, dendrites, and axons containing synapses and axon terminals from which neurotransmitters are released. A nerve is a group of neurons that serve a specific part of the body. A nerve may contain hundreds of neurons to hundreds of thousands of neurons. Nerves typically contain both afferent and efferent neurons. Afferent neurons carry signals back to the central nervous system and efferent neurons carry signals to the periphery. A group of neuronal cells at one location is called a ganglion. Electrical signals are conducted via neurons and nerves. Neurons release neurotransmitters at synapses (connections) with other nerves to allow for the persistence and modulation of electrical signals. In the periphery, synaptic transmission usually occurs in ganglia.

The electrical signals of the neurons are called action potentials. When the voltage potential across the cell membrane exceeds a certain threshold, an action potential is initiated. This action potential then propagates along the length of the neuron. The action potential of a nerve is complex and represents the sum of the action potentials of individual neurons therein. With the exception of the Langerhans' knot, myelinated neurons (and nerves) conduct in a hopping fashion. Without this hopping conduction, the electrical signal propagation would be quite slow (e.g., 2m/s in unmyelinated versus 200m/s in myelinated nerves).

The systems provided herein can provide energy pulses according to various stimulation parameters. For example, the stimulation parameters may include various stimulation time patterns, ranging from continuous to intermittent. During the signal on-time, energy is delivered at a frequency (e.g., 0.5Hz-30KHz, or in some embodiments, 0.5Hz-200Hz) for a period of time. The signal-on time is followed by a period of time during which no energy is transferred, referred to as the signal-off time. Neuromodulation patterns may include various combinations of pulse width (duration of a single pulse) and frequency (spacing between adjacent pulses). The neuromodulation pattern may be determined based on empirical evidence, e.g., based on patient data from previously treated patients, and/or may be customized for a particular patient. For example, the adjustment mode may be selected based on a mode determined to be successful for a patient with a similar clinical condition that is successfully treated. Modulation patterns can also be selected based on the record of neural activity in the patient to determine successful modulation and/or tracking of downstream effects of modulation (e.g., neuropeptide or neurotransmitter concentrations or other characteristics provided herein).

The duration of treatment can last as short as a few minutes to as long as several hours. The duration of treatment with the specified stimulation pattern may last for one hour. Pulse generation for neuromodulation is accomplished using a pulse generator. The pulse generator may use a conventional microprocessor and other standard electronic components. Stimulation parameters may also include frequency, duration, pulse shape, current or voltage parameters. The pulse generator for this embodiment can generate a pulse or energy signal having a frequency in the range of about 0.5Hz to 30KHz, a pulse width of about 10 to 1,000 microseconds, and a constant current between about 0.1 milliamp to 20 milliamps. The pulse generator may be capable of generating a ramp or ramp-up in current amplitude. In another embodiment, the pulse generator is a voltage generator that generates a constant voltage in the range of 0.5V to 10V. In certain embodiments, the pulse generator may be in communication with an external controller and/or monitor.

Bipolar stimulation of nerves can be achieved with multiple electrode assemblies, where one electrode serves as a positive node and the other electrode serves as a negative node. In this way, neural activation may be directed primarily in one direction (unilateral), such as efferent, or away from the central nervous system. Monopolar stimulation may also be performed. As used herein, monopolar stimulation includes a single electrode on the lead, while the implanted pulse generator itself or the ground electrode essentially serves as the second electrode remote from the lead electrode. With monopolar stimulation, a larger energy field is generated to electrically couple the electrode on the lead with the remote electrode. This allows nerve stimulation to be successfully performed using a single electrode placed only "in general proximity" to the nerve, which means that the spacing between the electrode and the nerve can be significantly greater than the "close proximity" required for bipolar stimulation. The magnitude of the allowable spacing between the electrode and the nerve will necessarily depend on the actual magnitude of the energy field that the operator generates with the lead electrode in order to couple with the remote electrode.

Techniques provided herein may include stimulating one or more lymphoid tissues. In addition, where the lymphatic tissue is innervated by multiple nerves, the stimulation may involve one or more nerves. For example, the electrodes may be positioned to deliver energy within a tissue region that affects multiple nerves.

Lymph node innervation

Provided herein are techniques for modulating neural pathways to produce therapeutic results. Modulation can be by direct electrical stimulation of the nerve/nerve pathway (i.e., an implanted stimulation device with electrodes) or by non-invasive means (i.e., by delivering energy from outside the body to the nerve to elicit action potentials or nerve activity, which can take many forms, including magnetic fields, external electric fields, or ultrasound). These stimulation techniques can also be used to modulate neural pathways to lymph nodes. That is, stimulation may target an upstream or downstream nerve location that is not within or adjacent to the lymphatic tissue, but is part of the neural pathway to and from the lymphatic tissue.

Lymphoid tissue may be innervated by the peripheral nervous system, which includes sensory and motor nerves. Such nerves may include the nerves of the autonomic nervous system, which carry signals to the glands, cardiac muscle and smooth muscle, and may be further divided into sympathetic and parasympathetic divisions. Adrenergic nerve fibers release neurotransmitters, such as adrenohormones (epinephrine), norepinephrine (noradrenaline) or dopamine. These neurotransmitters are released at synapses, which are junctions between the axons of one nerve cell and the dendrites of another nerve cell (or junctions/synapses with non-nerve cells). Sympathetic norepinephrine nerve fibers innervate certain lymphoid tissues and are usually introduced into the T lymphocyte region and the plasma cell region, rather than the nodal region or the B lymphocyte region. For example, in the thymus, noradrenergic fibers enter in the perivascular nerve bundles and plexus. In the spleen, noradrenergic fibers enter with the vasculature and are distributed along the central artery and associated periarterial lymphatic sheaths primarily in the white marrow. Fibers branch from the dense plexus around the central artery and enter the thin-walled tissue where they terminate in the region of lymphocytes and other cell types. In the lymph nodes, noradrenergic fibers enter the portal, travel along the vasculature and in the subcapsular plexus, and branch to the cortex and parenchyma in the cortical region where they terminate in lymphocytes. In the gut, the circular sac and the peyer's patch, the norepinephrine fibers enter the serosal surface, travel longitudinally with the intramuscular layers, turn radially into the tuberous plexus, pass directly through the thymus dependent area, and branch densely between lymphocytes, enterochromaffin cells and plasma cells in the interfornix region. In the bone marrow, noradrenergic fibers enter with the blood vessels, distribute deeply in the bone marrow on those blood vessels, and branch sparsely into the material of the bone marrow. Other types of nerve fibers that may be found include peptidergic fibers. The effects of neuromodulation may be affected by the type of nerve fiber being stimulated.

Therapeutic effects of lymphatic tissue stimulation

Many diseases are caused or exacerbated by defects in the signaling of the immune system. These include autoimmune diseases (in which immune cells are activated against self-antigens), inflammatory diseases (in which immune cells remain in a destructive or active state for a long period of time), and cancer (in which tumors can develop a protective immune cell spectrum, thereby eliminating the ability of the immune system to attack abnormal or cancerous cells). The control points for many of these processes are present in lymph nodes. Circulating immune cells often enter lymph nodes (passively or through active/targeted processes) and receive different signals/commands than surrounding tissue and blood. Other immune cells (and supporting cells) reside in lymph nodes and continuously secrete signals into the blood stream to affect cells outside the lymph chamber. As described above, neural stimulation and control of any of these immune cell populations can be used to treat a number of diseases. Electrical activation of postsynaptic sympathetic neurons results in the release of catecholamines (epinephrine, norepinephrine, dopamine). Peptidergic neurons can also release peptide-based neurotransmitters such as NPY, substance P and VIP. Parasympathetic nerve fibers can release other neurotransmitters, such as acetylcholine. As provided herein, neuromodulation techniques may be used to treat a subject having one of the diseases or disorders provided herein. However, it will be appreciated that the examples are non-limiting and that the techniques may be used to treat any subject in need of alteration of immune function.

As provided herein, neuromodulation of lymphoid tissue may result in immunomodulation and/or changes in the activity or function of the lymphoid tissue. It is understood that certain treatment outcomes may be associated with blocking effects, while other treatment outcomes may be associated with an increase in lymphatic activity. In certain embodiments, neuromodulation of the lymph node results in a regional enlargement of the lymph node relative to the contralateral lymph node. Enlargement or hypertrophy may be associated with changes in perilymphatic myocyte tone, long-term recruitment, and recombination of lymphatic vessels surrounding lymph nodes, and/or molecular changes at key barrier tissues, such as High Endothelial Venules (HEVs) within lymph nodes, including alterations in important transport proteins, such as aquaporins (water/fluid transport), or CCL21/CXCL13 secretion (cytochemokines). These changes may result in significant changes, including expansion, in cell density, cell count, and overall lymph node tissue environment. In addition, activation of lymph nodes can result in activation chains that expand or amplify local activation to systemically activate the lymphatic system. That is, local stimulation may result in both downstream and upstream activation of the lymphatic system. Thus, local neuromodulation may be used to activate a systemic immune response. Such activation may be beneficial for subjects with disorders associated with a wrong or reduced immune response. Such activation may also enhance the body's own response against pathogens. Thus, neuromodulation may be used to achieve altered lymph flow, altered immune cell trafficking into/out of lymphoid tissues, altered immune cell phenotype or local immune response, and/or antigen trafficking into/out of lymphoid tissues. Local stimulation may result in tissue or site-specific increases in the recruitment of lymph or immune cells.

Lymphoid tissue neuromodulation may be used to alter the immune cell population produced by the lymphoid structures. In one embodiment, neuromodulation of the lymph nodes may result in an increase in the population of lymphocytes circulating in the lymph fluid. Neuromodulation of lymph nodes will modulate the local concentration profiles of type 1 (pro-inflammatory) cytokines (e.g., IL-12, TNF- α, IFN- γ, IL-2, TNF- β) and type 2 (anti-inflammatory) cytokines (e.g., IL-4, IL-10, IL-13, IL-6). This modulation can occur by the release of noradrenaline from the nerves of lymphoid organs and its corresponding binding to the beta adrenergic receptors of T cells. In one embodiment, neuromodulation of the lymph nodes may result in an increase or decrease of B or T cells circulating on the lymph fluid, or an increase or decrease of B or T cells or dendritic cells recruited into the lymphoid tissue. Thus, neuromodulation of lymphoid tissue may lead to opportunities for patterns of cell migration. This migration pattern can be observed using in vivo bioluminescence imaging.

The subject's disorder can be used to select one or more appropriate lymphoid structures for neuromodulation. For example, occipital, auricular, cervical, axillary or intracorporeal epicondylar lymph nodes tend to enlarge in response to a particular pathogen, while other lymph nodes, such as the inguinal, pulmonary, mediastinal, intraabdominal lymph nodes, may be more susceptible to lesions associated with cancer or lymphoma.

In another embodiment, neuromodulation may be used to affect lymphatic drainage or flow. For example, stimulation with specific parameters may increase local and/or systemic lymphatic drainage, which in turn may enhance the resolution of the immune response. In one implementation, neuromodulation may enhance wound healing by enhancing drainage of pathogens away from the wound site and recruitment of additional immune cells against infection. Modulation of interstitial fluid flow in tumor or diseased tissue by neuromodulation of downstream lymphatic vessels may also be used to improve targeted delivery and local concentration of drugs. In another embodiment, neuromodulation at blocking frequencies (e.g., greater than 1000Hz) or voltages may be used to reduce neuronal input to lymph tissue.

An increase in drainage may also provide a benefit to subjects with a lymphatic circulatory disorder (such as lymphedema), such as a reduction in limb swelling. Lymphatic vessel flow is regulated by interstitial fluid pressure at the site of the primary lymphatic capillaries, the unidirectional valves of the lymphatic vessels, and the nerves and hormones that control the contraction of smooth muscle cells of the lymphatic and lymphatic structures. In addition to releasing neurotransmitters that amplify autonomic regulatory responses to increase lymphatic flow, neuromodulation may also activate local muscle contraction responses. For subjects with cancer, a reduction in lymphatic drainage/flow or cellular trafficking within the lymphatic system may help reduce the likelihood of metastasis. Such blocking parameters may include a relatively high stimulation voltage or frequency relative to the activation voltage or frequency.

Assessing lymphoid tissue function

The disclosed techniques can be used to assess lymphatic function. The disclosed techniques may use direct assessment of lymphatic condition or function. For example, for subjects in need of increased lymphatic drainage, such drainage can be monitored before, during, and/or after stimulation to determine whether a sufficient increase in the selected parameter has been achieved. Thus, lymphatic drainage can be assessed by one or more in vivo techniques for determining lymphatic drainage. In one embodiment, the exogenous contrast agent is administered directly to the lymphoid tissue or indirectly via intradermal injection. For example, gadolinium based contrast agents may be used. Depending on the desired clinical outcome, local or systemic blood flow may be addressed. For example, MR lymphangiography may be used to assess lymphatic drainage in a limb. However, in the case of subjects with implantable electrodes, MR imaging can be challenging.

In another embodiment, fluorescence micro-lymphangiography (FML) may also be used to assess lymphatic drainage. FML employs intradermal application of a fluorescent dye, which is FITC conjugated to dextran (FITC-dextran), and video fluorescence microscopy to obtain high resolution images. In another embodiment, quantum dot optical lymph imaging may be used for in vivo lymph imaging and lymph flow assessment. In another embodiment, imaging may include dyes or indicators targeting lymphoid specific markers, such as LYVE-1, Prox-1, pedigree protein, and VEGFR 3.

Images from the evaluation techniques may be received by the system for automatic or manual evaluation. Based on the image data, the stimulation parameters may also be modified. In one embodiment, the evaluation parameter is the size or estimated relative change in volume of the stimulated lymphatic tissue. For example, if the image data indicates that the lymph node size increases beyond a threshold (e.g., an estimated volume increase of at least 25% or 50% relative to the unstimulated state), then the stimulation may be considered successful and the parameters may be unmodified, or may step back to the lowest energy that achieves the desired result. Furthermore, the increase in size may be assessed within a predefined time window (e.g., within 5-10 minutes after the start of the adjustment).

Similarly, if lymphatic drainage increases in the presence of stable vital signs and other health indicators, the stimulation frequency or voltage may be stepped back to the lowest energy to achieve the desired result, e.g., to maintain the desired elevated lymphatic drainage. In other embodiments, lymphatic drainage or changes in size are used as markers of local neurotransmitter concentration, and as surrogate markers of local immune (immune interaction) cell exposure to phenotypically regulated neurotransmitters, and effectively as markers of predictive effect on immune function.

Additionally or alternatively, the system can assess the presence or concentration of neurotransmitters or cells in lymphatic tissue or fluid. Lymph fluid or tissue can be obtained by fine needle aspiration, and assessment of the presence or level of neurotransmitters (e.g., peptide transmitters, catecholamines) can be performed by any suitable technique. Analysis of secondary signaling molecules may also be useful, including inflammatory molecules and cytokines (i.e., TNF- α or IL6), whose secretion from local immune cells may be achieved through neuromodulation.

In another embodiment, a change in the type and/or number of cells in a lymph node or lymph tissue may be indicative of lymph tissue function. Cell populations can be assessed by ex vivo techniques such as flow cytometry. In another example, lymphocyte populations can be assessed by laser scanning In Vivo Confocal Microscopy (IVCM) using endogenous contrast. In the case where lymphoenlargement is mediated by cell entry into lymph nodes, a relative increase in cell population may also indicate such cell entry. Assessing cell recruitment and migration in the lymph nodes by measuring changes in lymph node size; ultrasound examination, cross-sectional CT or MRI can be used to assess size. These imaging assays may include morphological criteria to capture neuromodulation-induced lymph node structural changes. As described above, contrast enhanced MRI may be used. Contrast agents may be used dynamically, where an increase or decrease in contrast agent motor power may be indicative of a microcirculation change associated with neuromodulation (such as blood/lymph volume, microvascular permeability, or increased fractional volume of extracellular space within lymphatic tissue). Nanoparticle-enhanced MRI can also provide a method of assessing neuromodulation of lymphoid tissue. For example, superparamagnetic iron oxide (ferriotran-10) is known to enter lymphoid tissues and bind to macrophages, producing reduced signal intensity on T2 and T2-weighted images (image characteristics depend on cell density and tissue size). PET can be used to analyze the neuromodulatory effect on lymph tissue; F-FDG uptake can indicate increased glucose utilization in immune cells affected by neural stimulation. Other newer imaging agents can be used to analyze nerve stimulation-induced lymph node changes, including near infrared fluorescent probes recently used to visualize lymph nodes in animals following intravenous (pan-nodal) or subcutaneous (regional-nodal) administration.

Examples

Animal subjects were directly stimulated and stimulation data for neurotransmitters and three neuropeptides (with and without stimulation) were collected from lymph node tissue and from lymph fluid draining the node. Figure 13 depicts the popliteal lymph node on the right (stimulated) leg and left (unstimulated) leg after stimulation. In each subject, the stimulated lymph node (8) appeared larger than the "unstimulated" lymph node (6). In addition, stimulated lymph was shown to have an overall weight gain as compared to unstimulated lymph nodes, as shown in fig. 14.

Figure 14 shows the results of a comparison of stimulated versus unstimulated lymph nodes for different groups of subjects after stimulation of the popliteal lymph node and across different stimulation voltages. For each subject, the stimulated popliteal lymph node and the unstimulated contralateral lymph node were removed after stimulation, and the lymph node weight ratio was determined for each individual subject. In the control, the weights of stimulated and unstimulated lymph nodes were similar, with a ratio of 1. At certain stimulation voltages (0.5V, 2V, 5V and 7V), stimulated lymph nodes weigh almost twice as much as unstimulated lymph nodes. At even higher voltages (10V), this effect was not observed. The observed hypertrophy of stimulated lymph nodes may be associated with increased lymphatic activity, such as increased flow of fluid into the extracellular space of lymphatic tissue, or increased cellularity due to immune cell recruitment in stimulated lymph nodes.

Studies were also conducted to examine the effect of neuromodulation on the release of various neurotransmitters in both lymph nodes and lymph fluid after stimulation, with treatment group N-5-8. Figure 15A shows the concentration of epinephrine in the popliteal lymph nodes after stimulation at different voltages, and figure 15B shows the concentration of epinephrine in the lymph fluid after stimulation at different voltages. A significant increase was observed for some stimulation voltages (0.5V, 2V, 5V and 7V), while the effect was less pronounced at higher voltages (10V). For example, stimulation at 0.5V results in an increase in epinephrine concentration by more than 20-fold in both lymph nodes and lymph fluid.

Fig. 16A shows the concentration of noradrenaline in the popliteal lymph nodes after stimulation with different voltages, and fig. 16B shows the concentration of noradrenaline in the lymph fluid after stimulation with different voltages. For norepinephrine, a similar increase in release is observed in response to stimulation. Based on the samples observed, the effect drops at higher (10V) stimulation voltages.

Fig. 17A shows the concentration of dopamine in the popliteal lymph node after stimulation with different voltages, and fig. 17B shows the concentration of dopamine in the lymph fluid after stimulation with different voltages. Generally, dopamine showed the opposite trend (higher levels at higher applied voltages) and for most applied voltages, the dopamine concentration was not significantly different from the control.

Effects of neuromodulation on peptide transmitters were also observed. Fig. 18A shows the concentration of neuropeptide Y in the popliteal lymph node after stimulation at different voltages, and fig. 18B shows the concentration of neuropeptide Y in lymph fluid after stimulation at different voltages. Fig. 19A shows the concentration of substance P in the popliteal lymph node after stimulation at different voltages, and fig. 19B shows the concentration of substance P in the lymph fluid after stimulation at different voltages. Substance P did not show a significant increase in lymph nodes, but for some stimulation voltages, it was slightly elevated relative to controls in lymph fluid. Fig. 20A shows the concentration of vasoactive intestinal peptide in the popliteal lymph node after stimulation at different voltages, and fig. 20B shows the concentration of vasoactive intestinal peptide in the lymph fluid after stimulation at different voltages. It should be noted that stimulation at extreme frequencies (i.e. 30kHz) appears to have blocked the release of some neurotransmitters/neuropeptides.

Figure 21 shows a comparison of adrenal hormone concentration in stimulated and unstimulated (or contralateral left popliteal) lymph nodes in the same subject. One observed result is that stimulation appears to produce a contralateral stimulation effect. For example, at certain test voltages (0.5V, 2V, 5V and 7V), the contralateral lymph node was stimulated to release significantly more adrenal hormone than the control. This points to possible upstream and/or downstream connections. For example, a stimulus that produces increased local lymphatic activity may result in the release of a signal (e.g., neurotransmitter) that circulates through the lymphatic system and subsequently activates the contralateral lymph nodes. Such activation may also provide central or peripheral nervous system activation, which in turn may be used to further amplify the local activation effect through neural connections or reflexes. Fig. 22 shows a comparison of dopamine concentrations in stimulated and non-stimulated contralateral lymph nodes in the same subject. For dopamine, there was little change in contralateral effect compared to controls as observed on stimulated lymph nodes. Although these initial results did not elucidate the manner of connection between the stimulated and contralateral sides, it should be noted that the increased size difference between the stimulated and contralateral lymph could not be explained by neurotransmitter measurements (since no differential effect was observed). To examine the effect of nerve chain activation on promoting lymphatic function, a series of control studies were conducted to examine stimulation of intact nerves and severed nerves relative to intact, unstimulated controls. The severed nerve cannot complete nerve signal conduction through a direct nerve pathway to the lymph node, and a control is provided for a complete nerve experiment.

FIGS. 23A-F show the concentrations of various neurotransmitters in the lymph tissue of stimulated lymph nodes with intact nerves, stimulated lymph nodes with severed nerves, and controls. For epinephrine (fig. 23A) and norepinephrine (fig. 23B), intact nerves showed significantly greater neurotransmitter release relative to control and severed nerves. Dopamine levels (fig. 23C) were elevated in the controls relative to the controls and the severed nerves. A lesser degree of effect was observed with neuropeptide Y (fig. 23D). Substance P levels showed the only opposite effect (fig. 23E), with the highest level of severed nerves, likely as a result of wound-mediated release. VIP levels were relatively unchanged between samples (fig. 23F). Generally, these results indicate that changes in lymph node neurotransmitter and/or neuropeptide concentrations can be attributed to excitation/stimulation of neural pathways.

In addition to examining the effect of stimulation voltage on lymphatic activity, various stimulation frequencies were tested. FIGS. 24A-F show the concentration of various catecholamine neurotransmitters in lymphatic tissues at different stimulation frequencies. The greatest effect was observed with epinephrine (lymph node fig. 24A, lymph fluid fig. 24D) and norepinephrine (lymph node fig. 24B, lymph fluid fig. 24E), which had the greatest change relative to the control, and its peak concentration in both lymphoid tissue and lymph fluid was observed following stimulation at 20 Hz. For norepinephrine, stimulation at 20Hz appears to produce a significantly greater increase in release relative to other frequencies. For the three neurotransmitters examined, stimulation at 30KHz appeared to be associated with a lack of neurotransmitter release. The results for dopamine are shown in fig. 24C (lymph nodes) and fig. 24F (lymph fluid). FIGS. 25A-F show the concentrations of various peptide neurotransmitters in lymphoid tissues at different stimulation frequencies. The results for neuropeptide Y are shown in fig. 25A (lymph node) and fig. 25D (lymph fluid), for substance P in fig. 25B (lymph node) and fig. 25E (lymph fluid), and for vasoactive intestinal peptide in fig. 25C (lymph node) and fig. 25F (lymph fluid). The results indicate the frequency with which specific neurotransmitters or neuropeptides can be released into the lymph node compartment.

In addition to measuring neurotransmitters and neuropeptides associated with neural stimulation, the total cell number within the lymph nodes (and surrounding tissues/organs) is also measured as an initial measure of adaptive immune function (i.e. immune cell recruitment or capture in lymphoid tissues for increased antigen screening). Fig. 26 shows a comparison of cell count results from a directly stimulated lymph node, a contralateral ("unstimulated" or indirectly stimulated) lymph node, and a distal lymph node (axillary lymph node in the arm), while fig. 27 shows a comparison of cell count results for lymphoid tissue and blood in a subject, and fig. 28 shows a comparison of cell count results for spleen, thymus, and liver of a subject. The stimulation parameters for the results shown in fig. 26-28 were again 0.5V, 20Hz, for 5 minutes (pulse width 200 mus). Total white blood cell count (WBC) was measured, as well as specific cell counts of Neutrophils (NE), Lymphocytes (LV), Monocytes (MO), and Basophils (BA) for each subset of the white blood cell population. For the results shown in fig. 26-28, the leftmost bar in all figures represents the initial control subject, the middle bar in all figures represents the sham subject (electrode implanted but not stimulated), and the rightmost bar in all figures represents the stimulated subject. After only five minutes of stimulation in the directly stimulated lymph nodes, the number of lymphocytes increased dramatically, while other cell types were unchanged. This supports a lymphocyte-specific mechanism of capture or rapid recruitment of lymphocytes within lymph nodes via neuromodulation within a short time frame. Furthermore, antigen presenting cells such as monocytes do not show an increase in cell number over a five minute stimulation period (indicating that cells found primarily in the surrounding tissue but not in the blood do not respond to stimulation-induced recruitment or require longer stimulation/experimental time to respond). In contrast, the exact opposite effect was observed in the contralateral (or indirectly stimulated lymph nodes). That is, the number of all blood cell subtypes is significantly reduced in the opposing lymph node. Interestingly, no significant difference in catecholamine and neuropeptide levels was noted in the directly and indirectly stimulated lymph nodes. However, methods of measuring concentration throughout a lymph node do not account for differential changes in local concentration (i.e., around the nerve endings that innervate a particular lymph node region or chamber). In the distant axillary lymph nodes, there was no statistical change in the number of any cell types between the stimulated subjects and the controls. This suggests that systemic or global changes in neurotransmitter/neuropeptide levels are not responsible for differential regulation of immune cell migration/recruitment (i.e., adaptive immune function) in the two popliteal lymph nodes. This initial data represents the cross nerve reflex as depicted in fig. 4.

Furthermore, the number of cells within the lymph fluid is significantly increased (despite the increased number of lymphocytes in the directly stimulated lymph nodes). This suggests that the contralateral or indirect effects of neuromodulation of the adaptive immune reflex produce opposite responses at many nodes of the lymphatic system, resulting in opposite "release" of immune cells from many adjacent nodes (as compared to nodes recruited to local stimuli). However, again this effect is not shown as reaching the blood chamber within the five minute stimulation time point.

Figure 28 shows that neural networks and signaling may have been extended to other primary immune tissues/organs because specific cell types are recruited or released from the liver, spleen and thymus. Interestingly, monocytes appear to be preferentially trapped in the liver due to stimulation of the adaptive immune reflex, neutrophils and basophils appear to be preferentially recruited to the spleen, while lymphocytes are also released back into the circulation by the liver. This again suggests differential regulation between the different parts of the adaptive immune system (i.e. antigen presentation and cognate antigen screening) and the adaptive and innate immune systems and cell types.

Figure 29 shows the results of partial or complete excitation of axons based on electrode placement. The leftmost bar in the direct and contralateral data sets represents the control, the middle bar represents the normal electrode placed directly on the nerve, and the rightmost bar represents the gap or more distant (relative to direct contact) electrode placement. In the experiment, the electrodes were placed via insertion through a small incision in the skin (rather than complete surgical exposure of the nerve for placement). In one experiment, electrodes were inserted across the nerve (similar to full surgical placement). In another experiment, the electrodes were placed on one side of the nerve (with an additional gap between the electrode and the nerve). Controls were performed as in the previous experiment. Using the same stimulation parameters as in previous experiments (i.e., 0.5V, 20Hz, 200 μ s pulse width), electrodes placed across the nerve produced the expected response (i.e., direct stimulation of increased numbers of cells in the lymph nodes and decreased numbers of cells in the contralateral side). However, when the electrodes were placed with additional clearance between the electrodes and the nerve, a direct but contralateral reaction was observed. This demonstrates that neural pathways that provide control of direct and contralateral responses (e.g., efferent and afferent neural pathways) can achieve differential control of systemic immune function. In this case, the electrodes that span (and directly oppose) the nerve fire all axons within the nerve bundle, but the electrodes placed on one side (and with gaps) may fire only a portion/subset of those axons.

Figures 30 and 31 show that recruitment (non "draining") of lymphocytes by both the liver and spleen is also expected, as shown by the neuromodulation data provided herein. That is, based on previously published data, the demonstrated elimination of lymphocytes from the liver and spleen provided herein is an unexpected result. As shown herein, liver and spleen lymphocyte depletion mediated in response to neuromodulation may be the result of suppressing effects in the contralateral lymph nodes or the failure of traditional antigen injection to mimic the long loop reflex of the neural input necessary to trigger an adaptive immune reflex (as shown in fig. 9).

The disclosed neuromodulation techniques may be used in conjunction with neuromodulation systems. Fig. 32 is a schematic diagram of system 10 for neuromodulation, such as stimulation of nerves innervating lymphatic tissue. The depicted system includes an implanted electrode assembly 12 coupled to a pulse generator 14 via one or more leads. The electrode assembly 12 is configured to receive energy pulses via the leads, which in turn result in clinical effects at the electrode placement site. In certain embodiments, the pulse generator 14 may be implanted at a biocompatible site (e.g., the abdomen), and the one or more leads internally couple the electrode assembly 12 and the pulse generator 14. In certain embodiments, the electrode assembly 12 and/or the pulse generator 14 may communicate wirelessly, for example, with the controller 16, which may in turn provide instructions to the pulse generator 14. In other embodiments, the pulse generator 14 may be an external device, e.g., operable to apply energy transcutaneously or in a non-invasive manner, and in certain embodiments, may be integrated within the controller 16. In embodiments where the pulse generator 14 is implanted, the implantation site may be selected to reduce tension via the one or more leads. Once positioned to apply the energy pulse to the desired site, the system 10 may initiate neuromodulation to achieve the desired clinical effect. The stimulating includes stimulating at least one neuron innervating lymphatic tissue. The stimulation may include stimulation of sensory and/or efferent/effector nerve fibers.

In certain embodiments, the system 10 may include an evaluation device 20 that is coupled to the controller 16 and that evaluates the proxy characteristic indicating whether the adjustment objective has been achieved. For example, modulation may result in local lymphoid tissue or function changes, such as tissue structure changes, increased drainage, and the like. Modulation may also result in changes in immune function, such as changes in immune cell populations in the presence or concentration of compounds on lymphoid tissues. Based on the evaluation, the regulating parameters of the controller 16 can be changed. For example, if successful modulation is associated with an increase in lymph node size, the lack of observation of the size increase within a defined time window relative to the start of the procedure (e.g., 5 minutes, 30 minutes) may require an increase in frequency or voltage or other parameter, which in turn may be provided by the operator to the controller 16 to define the energy pulse of the pulse generator 14.

Fig. 33 is a block diagram of certain components of system 10. As provided herein, the system 10 for neuromodulation may include a pulse generator 14 adapted to generate pulses of energy for application to tissue or nerves of a subject. The pulse generator 14 may be implantable or may be integrated into an external device such as the controller 16. The controller 16 includes a processor 30 for controlling the system 10. Software codes or instructions are stored in the memory 32 of the controller 16 for execution by the processor 30 to control the various components of the device. The controller 16 and/or the pulse generator 14 may be connected to the electrode assembly 12 via one or more leads 33.

The controller 16 also includes a user interface having input/output circuitry 34 and a display 36 adapted to allow a clinician to provide selection inputs or stimulation parameters or more stimulation programs to treat and/or monitor a condition of the subject. Each stimulation program may include one or more sets of stimulation parameters including pulse amplitude, pulse width, pulse frequency, and the like. The pulse generator 14 modifies its internal parameters in response to control signals from the controller device 16 to change the stimulation characteristics of the energy pulses transmitted to the subject through the lead 33. Any suitable type of pulse generating circuit may be employed including constant current, constant voltage, multiple independent current or voltage sources, etc. The energy applied is a function of the current amplitude and the pulse width duration.

In one embodiment, the memory 32 stores different operating modes that may be selected by an operator. For example, the stored operating mode may include instructions for executing a set of stimulation parameters associated with a particular treatment and/or monitoring site. Different sites may have different associated stimulation parameters. The controller 16 may be configured to execute appropriate instructions based on the selection rather than having the operator manually enter the mode. In another embodiment, memory 32 stores operating modes for different types of therapy and/or monitoring. For example, activation of lymphatic tissue function may be associated with a stimulation voltage or frequency range that is different from the stimulation voltage or frequency range associated with inhibiting or blocking nerve output and/or lymphatic tissue function. In a particular example, the blocking frequency is in the range of at least 1kHz, while the activating frequency is less than 1 kHz.

In another embodiment, memory 32 stores a calibration or setting mode that allows for adjustment or modification of stimulation parameters to achieve a desired result. In one example, stimulation is started at a lower energy parameter (e.g., 0.5V or 0.5Hz) and is increased automatically or incrementally upon receiving operator input. In this way, the operator can observe the stimulation effect as the stimulation parameters change.

The controller 16 may also be configured to receive inputs related to lymphatic function as inputs to the selection of stimulation parameters. For example, when the imaging modality is used to assess lymphatic flow, the controller 16 may be configured to receive a calculated flow value. The stimulation parameters may be modified based on whether the flow value is above or below a threshold value. In another example, the controller 16 may receive input from one or more sensors configured to assess the concentration of molecules (e.g., peptides or catecholamines) released as a result of the stimulus. Based on the sensed concentration, the stimulation parameter may be modified.

In another implementation, a successful stimulation parameter set may also be stored by the controller 16. In this way, a subject specific parameter may be determined. Furthermore, the validity of these parameters may be evaluated over time. If a particular set of parameters becomes less effective over time, the subject may be desensitized to the activated pathway.

In the depicted example, the ultrasound device 20A includes an ultrasound probe 42 capable of acquiring image data of lymph tissue to assess changes in size. Ultrasound device 20A may include control circuitry for controlling ultrasound probe 42 and analyzing the acquired image data. Ultrasound device 20A may include additional hardware components such as a display 44, memory 46, and input/output devices 46. Although the depicted example is an ultrasound imaging apparatus 20A, the evaluation apparatus 20 may comprise other types of imaging apparatuses (e.g., invasive or non-invasive), or other types of imaging techniques, such as magnetic resonance imaging. Furthermore, the evaluation device 20 may comprise a non-invasive optical sensor and a monitoring device. In yet another example, the evaluation device 20 may be a flow cytometer that receives samples from the patient before and after modulation and determines whether a change in one or more cell populations has occurred as a result of neuromodulation.

Fig. 34 is a flow chart of a method 50 for stimulating immune tissue. In the method, at step 52, electrodes are positioned on or near lymphatic tissue or near a nerve of interest, and at step 54, a pulse generator applies a plurality of energy pulses to the tissue via the electrodes to stimulate neurons to modulate lymphatic or immune function of the lymphatic tissue. Then, at step 56, the effect of the stimulus is evaluated. For example, one or more direct or indirect assessments of the status of lymphatic or immune functions or conditions may be used. Based on the assessed lymphatic or immune function, the modulation parameters of one or more energy pulses may be modified at step 58 to achieve a desired clinical result. Additionally or alternatively, the function or condition of the stimulated nerve itself may also be used as a metric for determining the effectiveness of the modulation parameter.

Successful modulation, such as an increase in tissue structure size (e.g., lymph node size) or a change in the concentration of released molecules, e.g., relative to a baseline concentration prior to neuromodulation, can be assessed via measured clinical outcomes. In one embodiment, successful modulation may involve an increase in concentration above a threshold, e.g., an increase in concentration above 50%, 100%, 200%, 400%, 1000% relative to a baseline concentration. For blocking therapy, assessment may involve tracking the decrease in the concentration of the molecule over time, e.g., at least a 10%, 20%, 30%, 50% or 75% decrease in the molecule of interest. Furthermore, for certain subjects, successful blocking therapy may involve maintaining a relatively stable concentration of a particular molecule in the context of other clinical events that may tend to increase the molecule. That is, successful blockade may block the potential increase. The increase or decrease may be measured within a certain time window from the start of the treatment, e.g. within 5 minutes, within 30 minutes. In certain embodiments, the change in neuromodulation is an instruction to stop applying the energy pulse if it is determined that the neuromodulation was successful. In another embodiment, a parameter of neuromodulation is altered if neuromodulation is unsuccessful. For example, the change in the modulation parameter may be an increase in modulation frequency, such as a 10-100Hz step increase in frequency, and evaluating the desired characteristic until successful neuromodulation is achieved. In another implementation, the pulse width may be varied. In other embodiments, two or more parameters may be changed together. If neuromodulation is unsuccessful after the plurality of parameter changes, the position of the electrode may be changed.

In one embodiment, the assessment can be performed before and after stimulation to assess the change in lymphatic function due to stimulation. If the expected change in the state of the assessed lymphatic functional property is above or below a threshold value, appropriate modifications may be made to the adjustment parameters. For example, if a change in the characteristic relative to a threshold is associated with successful activation of lymphatic tissue, the energy applied during neuromodulation may step back to a minimum level that supports the desired result. If the change in the characteristic relative to the threshold is associated with insufficient activation of the lymphatic tissue, certain modulation parameters may be changed, such as modulation voltage or frequency, pulse shape, stimulation pattern, and/or stimulation location. It should also be understood that certain desired clinical outcomes may alternatively be associated with blocking activation. In such embodiments, the assessment of reduced neurological and/or lymphatic function is associated with maintaining the regulatory parameter, and the regulatory parameter may be modified if an undesirable level of lymphatic activity persists.

Further, the assessed characteristic or condition may be a value or index (e.g., flow, concentration, cell population), which in turn may be analyzed by any suitable technique. For example, relative changes that exceed a threshold may be used to determine whether the tuning parameters are modified.

Fig. 35 is an overview of various lymphoid tissue locations in vivo, showing the neurostimulation effect of the immune structures. The effect may then be evaluated to adjust one or more parameters of the stimulus. As discussed herein, stimulation may be assessed via one or more assessment techniques. Figure 36 is an image of a stimulated popliteal lymph node 70 labeled with a dye. Such markers can be used to assess in vivo dimensional changes before, during and/or after modulation.

In one embodiment, ultrasound imaging is used to assess lymph node size. Figure 37 shows an ultrasound image of a subject's directly stimulated popliteal lymph node (as described above) prior to electrode implantation and neuromodulation. The lymph node size (0.17cm) measured using the ultrasound system interface closely matched the size of the excised unstimulated lymph node in FIG. 7. Sizing is performed by summarizing the changes in ultrasound contrast within the popliteal fat pad associated with the lymphatic structure. FIG. 38 shows images and measurements of the same popliteal lymph node after the above 5 minute stimulation (i.e., 20Hz, 0.5V, 200 μ s); the size increased to 0.3cm after stimulation. This data suggests that neurotransmitter release by lymphatics and immunomodulation produces a clear and measurable effect on tissue architecture that can be monitored using non-invasive imaging techniques.

Figure 39 is a graph of the fluorescence signal associated with known β -endorphin standards used to calibrate the experimental results. The graph of signal intensity (y-axis) versus concentration of standard (x-axis) shows the results of the competition assay. Thus, a lower fluorescence signal correlates with a higher concentration of β -endorphin. Figure 40 is a graph of results from animal studies showing that lymphatic stimulation drives an increase in β endorphin as measured in interstitial fluid of the spleen or blood of stimulated subjects. In one embodiment, the stimulation triggers the release of beta endorphin from immune cell stores, which in turn is detectable in interstitial fluid or blood samples. The stimulated samples were associated with lower fluorescence signals indicating higher β endorphin concentrations. Thus, the effectiveness of stimulation can be assessed via blood concentration measurements of immunologically active surrogate markers (such as β endorphin). Furthermore, as provided herein, neuromodulation may be associated with an increase in endogenous opioid concentration, and in turn, may be associated with a therapeutic benefit of alleviating pain.

Technical effects of the present disclosure include stimulating the adaptive immune reflex pathway via neuromodulation to produce a differential local physiological or immune change. For example, the disclosed technology allows for stimulation of lymphoid tissues that would be challenging to target via systemic techniques (such as drug therapy). In addition, the disclosed technology can be used to treat subjects with a variety of clinical conditions, including patients with lymphoid disorders, cancer patients, patients in need of immune modulation, and the like. The techniques may also be used as supportive treatment tools. For example, neuromodulation of lymphoid tissue and local recruitment of cells may be used to localize cells injected into a patient for cell-based therapy (e.g., DC-based cancer immunotherapy), or to limit metastatic spread of a primary tumor during chemotherapy. More targeted impact on the adaptive immune system can also be achieved by further localizing the neural stimulation signals (i.e., placing the electrodes closer to the local lymphatic target), differential excitation of afferent and efferent components of the neural pathway, and/or blocking of excitatory signals by applying a blocking stimulus (i.e., a high frequency stimulus) upstream of the stimulation electrodes.

The specific embodiments described above have been shown by way of example, and it should be understood that these embodiments may be susceptible to various modifications and alternative forms. It should be further understood that the claims are not intended to be limited to the particular forms disclosed, but rather to cover all modifications, equivalents, and alternatives falling within the spirit and scope of this disclosure.

Claims

1. A system for neuromodulating lymphatic tissue, comprising:

an electrode assembly configured to apply one or more energy pulses to a neuron of a subject to deliver energy to the neuron to neuromodulate lymph tissue in response to application of the one or more energy pulses;

a pulse generator configured to generate the one or more energy pulses;

a controller configured to cause the pulse generator to generate the one or more energy pulses in accordance with a stimulation parameter to cause the lymphatic tissue to be neuromodulated; and

a non-invasive evaluation apparatus configured to acquire image data of the lymphatic tissue and to identify a change in a characteristic of the lymphatic tissue based on the image data, wherein the controller is configured to modify the stimulation parameter based on the identified change in the characteristic.

2. The system of claim 1, wherein the stimulation parameters of the one or more energy pulses are selected such that immune function is modulated to result in an increase in the number of cells in the population of cells in the lymphoid tissue.

3. The system of claim 2, wherein the stimulation parameters of the one or more energy pulses are selected such that the immune function is modulated to result in a reduction in the number of cells in a cell population in the contralateral lymphoid tissue.

4. The system of claim 1, wherein the lymphatic tissue is a lymph node.

5. The system of claim 1, wherein the electrode is in direct contact with the neuron.

6. The system of claim 1, wherein the electrode is not in direct contact with the neuron.

7. The system of claim 1, wherein the stimulation parameters of the one or more energy pulses are selected such that immune function is modulated to result in no change in cell number in the population of cells in the contralateral lymphoid tissue.

8. The system of claim 1, wherein the stimulation parameters cause the electrode assembly to apply the one or more energy pulses at an energy in a range of 0.5V to 10V.

9. The system of claim 1, wherein the stimulation parameters cause the electrode assembly to apply the one or more energy pulses at a stimulation frequency in a range of 0.5Hz to 200 Hz.

10. The system of claim 1, wherein the neuron is a parasympathetic or sympathetic neuron innervating a primary, secondary or tertiary lymphoid tissue structure or transmission tissue.

11. The system of claim 1, wherein the subject is a subject diagnosed as having an autoimmune disease.

12. The system of claim 1, wherein the subject has one or more symptoms of tissue swelling, a need to alter fluid content of the tissue, a need to locally contain antigen, infection, foreign or host cells, a need to locally recruit immune cells, a need to locally remove specific immune cells, a need to alter a phenotype, activity or immune response of immune cells within lymph and surrounding tissues, or a need to alter the phenotype, activity or immune response of cells within the lymph and surrounding tissues.

13. The system of claim 1, wherein a change in immune function due to the neuromodulation is detectable in the subject within 30 minutes of applying the one or more energy pulses.

14. The system of claim 13, wherein a change in immune function due to the neuromodulation is detectable in the subject within five minutes of applying the one or more energy pulses.

15. The system of claim 13, wherein the detectable change is an increase in white blood cells in the lymphoid tissue relative to a baseline prior to application of the one or more energy pulses.

16. The system of claim 13, wherein the detectable change is an increase in white blood cells in lymph fluid from the lymphoid tissue relative to a baseline prior to application of the one or more energy pulses.

17. A system according to claim 13, wherein the detectable change is a decrease in white blood cells in the contralateral lymphoid tissue relative to a baseline prior to application of the one or more energy pulses.

18. The system of claim 13, wherein the detectable change is an increase in monocytes in the liver relative to a baseline prior to application of the one or more energy pulses.

19. A neuromodulation system, comprising:

an electrode assembly configured to apply one or more energy pulses to a neuron of a subject to deliver sufficient energy to the neuron to modulate immune function of lymphoid tissue such that the concentration of norepinephrine or epinephrine in the lymphoid tissue or fluid is increased by at least 100% relative to a pre-stimulation baseline in response to the one or more energy pulses, wherein the one or more energy pulses are applied at an energy ranging from 0.5V to 10V;

a controller configured to cause the one or more energy pulses to be generated; and

a non-invasive evaluation device configured to acquire image data of the lymphatic tissue during or after the delivery of the energy to the neuron, wherein the controller is configured to modify one or more parameters of the one or more energy pulses based on the image data.

20. The system of claim 19, wherein the neuron is a parasympathetic or sympathetic neuron innervating primary, secondary or tertiary lymphoid tissue structures or transmission tissue.

21. The system of claim 19, wherein the subject has one or more symptoms of tissue swelling, requires alteration of fluid content of the tissue, requires local inclusion of antigen, infection, foreign or host cells, requires local immune cell recruitment, requires local removal of specific immune cells, requires alteration of a phenotype, activity or immune response of immune cells within lymph and surrounding tissues, or requires alteration of the phenotype, activity or immune response of cells within the lymph and surrounding tissues.

22. The system of claim 19, wherein the one or more energy pulses are applied according to a stimulation parameter associated with neurotransmitter or neuropeptide release.

23. The system of claim 19, wherein the increase in concentration is measured in a lymphatic tissue sample.

24. The system of claim 19, wherein the increase in concentration is measured in a lymph fluid sample.

25. The system of claim 19, wherein the increase in concentration is an increase in concentration of a neurotransmitter or neuropeptide of at least 200% relative to the baseline concentration.

26. The system of claim 19, wherein the increase in concentration is an increase in concentration of a neurotransmitter or neuropeptide of at least 400% relative to the baseline concentration.

27. The system of claim 19, comprising an evaluation device configured to evaluate the increase in concentration.

28. A closed-loop neuromodulation system, comprising:

a pulse generator configured to apply one or more energy pulses to neurons innervating lymphatic tissue in accordance with one or more stimulation parameters of at least one of the one or more energy pulses to modulate lymphatic function of the lymphatic tissue;

a non-invasive assessment apparatus configured to receive information related to a condition or function of the lymphatic tissue, wherein the non-invasive assessment apparatus is configured to acquire image data, and wherein the information related to the condition or function of the lymphatic tissue is based on the image data; and

a controller configured to control the pulse generator to apply the one or more energy pulses according to the one or more stimulation parameters and to change the one or more stimulation parameters based on the information.

29. The system of claim 28, wherein the information relates to a change in lymphatic drainage.

30. The system of claim 28, wherein the controller is configured to vary a frequency of the one or more energy pulses based on the information.

31. The system of claim 28, wherein the controller is configured to vary the voltage of the one or more energy pulses based on the information.

32. The system of claim 28, wherein the stimulation parameter is based on a location of the lymphatic tissue.

33. The system of claim 28, wherein the pulse generator is configured to apply the one or more energy pulses to the neurons innervating lymphatic tissue via an electrode assembly, and the electrode assembly applies electrical energy.

34. The system of claim 28, wherein the pulse generator is configured to apply the one or more energy pulses to the neurons innervating lymphatic tissue via an electrode assembly, and the electrode assembly applies magnetic energy.

35. The system of claim 28, wherein the pulse generator is configured to apply the one or more energy pulses to the neurons innervating lymphatic tissue via an electrode assembly, and the electrode assembly is configured to be positioned on or in the lymphatic tissue.

36. The system of claim 28, wherein the pulse generator is configured to apply the one or more energy pulses to the neurons innervating lymphatic tissue via an electrode assembly, and the electrode assembly is configured to be positioned subcutaneously.

37. A neuromodulation system, comprising:

a pulse generator configured to deliver one or more energy pulses to the electrodes to cause a stimulating activity of the lymphoid tissue according to the mode of operation;

a non-invasive evaluation device configured to acquire image data of the lymphoid tissue and identify a change in a characteristic of the lymphocytes based on the image data; and

a controller configured to:

receiving one or more user inputs selecting the mode of operation for delivering the one or more energy pulses to the electrode to stimulate activity of the lymphatic tissue;

receiving one or more inputs related to the stimulatory activity of the lymphoid tissue; and

changing a parameter of at least one of the operating mode or modifying the one or more energy pulses based on the one or more inputs and the identified change in the characteristic.

38. The system of claim 37, wherein the one or more inputs related to the stimulatory activity indicate a change in fluid drainage.

39. The system of claim 38, wherein the change is an increase in fluid drainage.

40. The system of claim 37, wherein the one or more inputs related to the stimulatory activity are indicative of a change in a measured count of leukocytes, neutrophils, lymphocytes, monocytes, or basophils in the lymphoid tissue or fluid.

41. The system of claim 40, in which the change in the measurement count is an increase in the measurement count.

42. The system of claim 37, wherein the one or more inputs related to the stimulatory activity indicate a change in noradrenaline concentration in the lymphoid tissue or fluid.

43. The system of claim 42, wherein the change in concentration is an increase in the concentration.

44. The system of claim 37, wherein the one or more inputs related to the stimulatory activity are indicative of a change in adrenal hormone concentration in the lymphatic tissue or fluid.

45. The system of claim 44, wherein the change in concentration is an increase in the concentration.

46. The system of claim 37, wherein the one or more inputs related to the stimulatory activity are indicative of a change in neuropeptide Y concentration in the lymphoid tissue or fluid.

47. The system of claim 46, wherein the change in concentration is an increase in the concentration.

48. The system of claim 37, wherein the one or more inputs related to the stimulatory activity are indicative of a change in concentration of substance P in the lymphatic tissue or lymph fluid.

49. The system of claim 48, wherein the change in concentration is an increase in the concentration.

50. The system of claim 37, wherein the one or more inputs related to the stimulatory activity are indicative of a change in vasoactive intestinal peptide concentration in the lymphoid tissue or fluid.

51. The system of claim 50, wherein the change in concentration is an increase in the concentration.

52. The system of claim 37, wherein the one or more inputs related to the stimulatory activity indicate a size of the lymphatic tissue relative to a baseline size after application of the one or more energy pulses.